Pseudomonas aeruginosa is a ubiquitous Gram-negative opportunistic pathogen of compromised patients. Especially susceptible are individuals inflicted with burn wounds, eye complications, and cystic fibrosis. P. aeruginosa pathogenesis is facilitated by growth in hospital environments and its intrinsic resistant to antibiotics. P. aeruginosa produces a number of virulence determinants, which are either cell-surface components or secreted. P. aeruginosa produces four type-III secreted cytotoxins (ExoS, ExoT, ExoU, and ExoY), which inactivate the host innate immune system, the first line of defense against bacterial infection. The goals of this proposal are to define the molecular properties of the P. aeruginosa type-III cytotoxins ExoS and ExoT. ExoS and ExoT are bi-functional cytotoxins: the amino terminus of ExoS and ExoT inactivate Rho GTPases by acting as RhoGTPase Activating Proteins (RhoGAPs) to disassemble the actin cytoskeleton and inhibit internalization of P. aeruginosa by mammalian cells, while the carboxyl terminus of ExoS and ExoT comprises an ADPribosyltransferase domain. ExoS and ExoT ADP-ribosylate unique subsets of host proteins in mammalian cells. The targets of ExoS and ExoT control numerous cellular processes, including wound healing, tissue regeneration, and phagocytosis. This proposal addresses the basis for the physiological actions of ExoS and ExoT. Utilizing complementary molecular, cellular, and biochemical approaches, the goals of this application are to: characterize the ADP-ribosylation of Crk by ExoT; determine how ExoT and ExoS recognize their substrates for ADP-ribosylation, characterize the in vivo ADP-ribosyltransferase activity of ExoS, characterize the RhoGAP domain of ExoS, and measure the intracellular targeting of type-III cytotoxins in mammalian cells. Addressing these aims will define how ExoS and ExoT allow P. aeruginosa to establish infection in mammalian cells and define new strategies to detect, prevent, and control infection by this opportunistic pathogen.